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Altered enzyme heralds greener chemistry

MAKING “designer” enzymes – altered versions of nature’s own caralysts – may be simpler than scientists had thought. Chemists at Oxford University have made an enzyme work on a different chemical from its usual substrate by changing just one of the several hundred amino acids from which the enzyme is built. The technique could lead to the development of cleaner ways of making drugs and other useful chemicals.

Sabine Flitsch, Luet-Lok Wong and colleagues worked on an enzyme called cytochrome P-450, produced by a strain of the bacterium Pseudomonas putida. The microorganism uses the enzyme to add a hydroxyl (OH) group to camphor, a substance used in the manufacture of linaments. This reaction is essential to the bacterium’s metabolism. The chemists, however, have now altered the enzyme so that it adds a hydroxyl group to a completely different molecule called diphenylmethane, one of the compounds from which polyurethane resins are made. They believe their success will allow them to learn how to “tune” the enzyme to process other useful compounds in the same way.

Enzymes have been used in industrial chemical processes for years. The enzymes made by yeasts, for instance, are used to convert plant sugars into alcohol in fermentation. Chemists, however, would like to make designer versions of enzymes for industry, because they have many advantages over other chemical catalysts (see “The greening of chemistry”, New Scientist, 21 April 1990). They produce bigger yields, can speed up some reactions to millions of times their uncatalysed rate, and produce few or no waste products. Enzymes are also greener than most industrial catalysts because they normally work best at atmospheric pressure and at only a few degrees above room temperature. Many industrial catalysts need high pressures and temperatures to work efficiently, thereby wasting large amounts of energy.

To make their designer enzyme, the Oxford researchers concentrated on a small part of the molecule called its active site, which is the region that usually binds to camphor. The team suspected that subtly changing the active site would dramatically alter the type of molecule that would fit inside. First, they used a sophisticated computer program to predict what would happen if they substituted another amino acid for one of three amino acids lining the active site. These three amino acids all contain rings of six carbon atoms called aromatic groups and are the anchor points for the camphor molecule in the unaltered enzyme. The computer simulation indicated that even this subtle change would reduce the enzyme’s ability to bind with camphor, and increase its affinity for hydrophobic – water-repelling – molecules such as diphenylmethane.

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To test their ideas in the real world, the chemists turned to the tools of genetic engineering. They made a strand of DNA containing the gene sequence for the cytochrome enzyme, and altered this sequence so that it would instead code for a version of the enzyme carrying the substitute amino acid at its active site. They then inserted the engineered DNA into another bacterium in order to produce enough of the designer enzyme for them to study.

Flitsch, Wong and their team found that the enzyme was, as they had anticipated, very reluctant to hold onto camphor and instead bound to the hydrophobic diphenylmethane. They also found that the enzyme had processed the diphenylmethane in a similar way to the unaltered enzyme’s action on camphor – it had added a hydroxyl group to the molecule (Journal of the Chemical Society&colon; Chemical Communications, 1994, p 2761).

Although other chemists have altered enzymes so that they work on different molecules, this is the first time that researchers have achieved such a change by altering just one amino acid. And the fact that the Oxford researchers have achieved this with cytochrome P.450, which adds a hydroxyl group to the molecule that fits in its active site, is particularly relevant for the chemical industry. The hydroxylation reaction is an important first step in converting hydrocarbons derived from crude oil into more useful products such as drugs and agricultural chemicals. The team hopes that by tweaking the active site of their designer enzyme they can make it bind other hydrocarbons and so tailor it to specific starting materials and products.